PROCESS OF ISOLATING NATIVE ALBUMIN AND/OR GLOBULIN FROM AN AQUEOUS SYSTEM COMPRISING RAISING THE pH TO ABOVE 8.5 AND THEN LOWERING THE pH TO THE ISOELECTRIC POINT TO PRECIPITATE PROTEIN

ABSTRACT

A process for isolating from an aqueous solution in their natural form proteins of the class of albumin, globulin and mixtures thereof which includes raising the pH of the aqueous protein solution to at least 8.5 and then flocculating the protein at its iso-electric point.

finite Sates tent Brouwer et al. Aug. 29, 1972 [54] PROCESS OF ISOLATINGNATIVE 669,361 3/1901 Deycke ..260/122 ALBUMIN AND/OR GLOBULIN FROM1,428,820 9/1922 Thomson ..260/ 122 AN AQUEOUS SYSTEM COMPRISING2,219,791 10/1940 Rabald et al ..260/112 RAISING THE PH T0 ABOVE 35 AND2,391,559 12/ 1945 Faulkner ..260/122 X H N LOWERING THE PH TO THE2,460,550 2/1949 Strumia et a1. ..260/112 ISOELECTRIC POINT 0 2,684,9607/1954 Taylor ..260/ 123.5 et a] X 3,018,280 l/1962 Salzberg et a1..260/123.5 X [72] Inventors: Abraham Brouwer, Harderwijk;

Gerardus M. A. M. VanLoom, OTHER U L CA' ONS Reeuwllk, both NetherlandsJ. Am. Chem. Society, vol. 48, 1926, 763- 768, [73] Assignee:Melkcentrale Gouda N.V. l g a l -d E Ch v 1 3l 9 9 n ustri an ngineeringemistry, o 1 3 [221 Flled- A 1970 Smith et al., 211., pp. 1284- 1288[21] Appl. No.: 30,601 Chem. Abstracts, Vol. 44, 1950, 5416 P-1,Cammarata et a1.

[30] Foreign Application Priority Data Primary Examiner-Howard E. SchainApril 22, 1969 Netherlands ..69.06198 At n y-F pp & Jacobson [52] U.S.Cl. ..260/122, 260/112 B, 260/112 R [57] ABSTRACT [51] Int. Cl. ..C07g7/00 A process for lsolatlng from an aqueous solut1on in [58] Field ofSearch ..260/112, 112 B, 122 their n amt a1 form proteins of the classof albumin globulin and mixtures thereof which includes-raising [56]References cued the pH of the aqueous protein solution to at least 8.5UNITED STATES PATENTS and then flocculating the protein at itsiso-electric t. 2,450,810 10/1948 Opper et al ..260/112 pom 11/1969Liggett ..260/123.5

8 Claims, N0 Drawings The invention relates to a process of isolatingnative albumin and/or globulin from an aqueous system, such as whey andblood, by first of all raising the pH of the system and subsequentlyflocculating the protein at the isoelectric point.

From the Dutch Patent Application No.293,320 there is already known aprocess at which at room temperature, and with due regard to somecation-anion interaction conditions, the pH of an acid whey with the aidof a weak ion exchanger, such as Montmorillonite, is steadily increasedfrom 4 to 7 and immediately thereupon to 8 by adding small quantities ofcalcium hydroxide, which causes various protein fractions to beprecipitated. However, a disadvantage of said method is, that themanufacturing costs of the resultant protein fractions aredisproportionate to those of the proteins that are on the marketalready.

From the Dutch Patent Application No. 254,554 there is known anotherprocess in which whey is first of all evaporated under vacuum andimmediately thereupon concentrated to a total solids content of 80-85percent by weight by injecting steam. Although the resultant product iswatersoluble, this process has still the disadvantage that the saltcontent of the resultant whey paste is too high, as a result of whichthe use thereof is very limited.

In the German Patent Specification No. 830,153 there has been discloseda process of processing whey by adding to each liter of whey 0.2 to 2 gof iron, in the form of iron compounds, such as ferric chloride, andisolating the resultant precipitate containing albumin and phosphoricacid of the whey.

According to the process disclosed in the German Patent SpecificationNo. 835,982, albumins and phosphates are also precipitated from whey byadding an iron salt, but moreover the pH is also raised to about 8. Adisadvantage of said processes, in which iron salts are used toprecipitate the albumins, is, naturally, that the iron remains in theproduct in the form of the hydroxide, because of which the natural whiteof the proteins is detrimentally affected.

The British Patent Specification No. 1,045,860 discloses a process inwhich albumins are precipitated by adding polyphosphates, such as sodiumor potassiummetaphosphate, as lactalbumin phosphates.

The German Patent Specification No. 810,687 discloses a process in whichthe pH of acid whey is first of all raised to 7-7.5, subsequently it isheated to 100 C and immediately cooled to 40 C, whereas the pH isdecreased to the isoelectric point of albumin. A disadvantage of saidprocess is that the resultant proteins are denatured by the heatingprocess.

It was found now that albumins and/or globulins, without any of theproblems associated with the process of the German Patent SpecificationNo. 810,687, may be isolated in native form from an aqueous system likewhey and blood by raising the pH of the system to at least 8.5 by addingphysiologically tolerable basic compounds at a temperature below thedenaturing temperature of albumins and/or globulins, immediatelythereupon decreasing the pH to the isoelectric point of the albuminsand/or globulins (i 0.1) by adding an acid at a temperature below thedenaturing temperature of albumins and/or globulins, separating off theprecipitated proteins, and, if desired, repeating the entire processonce or more than once. Owing to this variation in pH values, thelinkage between the protein, the anions and cations is broken orweakened, causing a decrease of the solubility of the protein at itsisoelectric point. The process is generally carried out at a temperatureof from 40 till 50 C.

If whey is used as a starting material, preferably a mixture of equalparts of water and the liquor, which remains after the lactose has beenremoved from whey which is evaporated to a solids content of 60 percentby weight, is used. This liquor is obtained by separating off thecrystallized lactose by passing it through a centrifuge. The resultantliquor contains 10-12 percent by weight of albumins globulins. Afterdilution with an equal quantity of water there is thus obtained astarting material containing 5-6 percent by weight of albumin globulin.

Another proper starting material is aqueous solution of whey powder.

When blood and blood serum (blood passed through a centrifuge) are usedas the starting material, it is also preferable to dilute it with equalquantities of water before heating and rendering alkaline.

When blood is used as the starting material, the process is lesscomplicated than when whey is used as the starting material, because thesalt content of blood is lower than that of whey. The adjustment of thepH of at least 8.5 is preferably made at a temperature of 45 C. Allphysiologically tolerable bases, such as NaOH, KOH, and Ca(OH may beused for said purpose. However, 33 percent sodium hydroxide ispreferably used. The pH of 9 may be liberally exceeded, though the pH ispreferably adjusted to 9.5 10.5. The adjustment of the pH for theisoelectric point may be made at any temperature below the denaturingtemperature of albumins and/or globulins, though a temperature rangingfrom 40-50 C is preferably used. There are no specific demands made onthe acid to be used, apart from the physiological tolerance. Suitableacids are, for instance, HCl, H 50 H PO and organic acids, in particularcitric acid.

The pH is preferably accurately adjusted to the isoelectric point, whichis between 4.0 5.2 depending on the salt content. When the pH values aretoo low, the protein will start to dissolve again. pH values that arelower than those corresponding with the isoelectric point shouldtherefore be avoided. Albumins and/or globulins will start to flocculateas soon as the acid is added, because there will be a sufficient, localconcentration of acid to invoke flocculation. These local, relativelyhigh concentrations can occur in their return because stirring has to bedone at slow speed, so as not to damage the structure of the resultantfloccules. There are no serious objections to this local flocculation,however, because the resultant flocculation is entirely in accordancewith the object of the invention. When the flocculating process of thealbumins and/or globulins has been completed, they may be isolated byconventional methods, such as by being passed through a centrifuge witha great force of gravitation, and air dried. In many cases the saltcontent of the albumin and/or globulin will be higher than is desirablefor food purposes. In these cases the isolated product at roomtemperature is redissolved in water rendered alkaline until a solutionis obtained containing 5-6 percent by weight of albumin and/or globulin,the pH is raised to at least 8.5 by adding physiologically tolerablebasic compounds at a temperature below the denaturing temperature of thealbumin and/or globulin, the pH is immediately thereupon decreased tothe isoelectric point (i- 0.1) by adding an acid at a temperature belowthe denaturing temperature of albumin and/or globulin, the precipitatedproteins are separated off, and, if desired, the entire process isrepeated once or more than once. The final product is soluble in neutralor slightly basic medium.

EXAMPLE I A mixture of liters of pig's blood and 10 liters of water washeated to 40 C in a round-bottomed flask in a water bath. 33 percentsodium hydroxide was then added to pH=l0. 35 percent hydrochloric acidwas then immediately added until the mixture had a pH of 5.1. Thealbumin and globulin then started to flocculate. The entire mixture waspassed through a centrifuge, which yielded a still slightly coloredprotein percipitate. This was dispersed in water with pH=8. The wholeprocess was then repeated. The resultant product, after having been airdried for 24 hours, was 90 percent pure.

EXAMPLE II 10 liters of evaporated whey having a solids content of 30percent by weight was heated to 41 C. 33 percent sodium hydroxide wasthen added to pH=1 1.5. 35 percent hydrochloric acid was immediatelythereupon added with slow stirring to pHflD. The albumins and globulinsstarted to flocculate and could be concentrated with the aid of acentrifuge. The centrifugal cake was suspended in water to a proteinconcentration of 5 percent by weight. The dispersion was then heated to41 C and dissolved with 30 percent sodium hydroxide at pH=9. By adding35 percent hydrochloric acid the proteins were re-precipitated atpl-i=4.l. The resultant flocculated protein was concentrated by means ofa centrifuge and suspended in water to a solids content of percent byweight. The resultant protein was dissolved in a mixture of water andpercent sodium hydroxide, with pH=8.5, and the resultant mixture wasthen dried in a spray drier at temperatures up to 80 C. The finalproduct contained 90 percent by weight of protein.

EXAMPLE III In a beaker having a content of 15 liters there were admixed4 liters of liquor, having a protein content of 10 percent by weight,and 6 liters of water. The mixture was heated with stirring to 46 C,while special care was taken that there was no local overheating and noair was whipped into it. To this mixture was added 30 percent sodiumhydroxide with vigorous stirring until the pH was 10.5. After 2 minutes,with slow stirring, and by dding 35 percent hydrochloric acid th pH wasecreased o 4.1. he protein coagu ated and precipitated slowly as a whitefloccular mass. When the above-mentioned clear liquid had been decanted,the protein was first of all passed through a centrifuge and then takenup in water again, the pH of the water had been adjusted to pH=4.l withhydrochloric acid. After having been stirred and optionally decanted,the liquid was passed through the centrifuge again, and the protein wasthen taken up in water and dissolved in water, that had been adjusted topH=8.5 with NaOH, with vigorous stirring. After reprecipitation withacetic acid at pH=4.l the liquid was passed through the centrifugeagain. The resultant protein with stirring was dissolved in water havingpH=8 to as great a concentration as was possible. The resultant solutionwas dried in a spray drier having an air temperature of C. The resultantproduct was water-soluble and had the following analytical values:

86 percent by weight of protein 5 percent by weight of moisture a traceof lactose 5 percent by weight of ashes some percentages of organicsalts.

It is to be understood that the above examples are only for purposes ofillustration and are in no way to limit the invention thereto.Furthermore, this invention can be practiced using other similarcompounds and reactions without departing from the scope of theinvention as set forth in the appended claims.

What is claimed:

1. A process for isolating a protein in its natural form selected fromthe group consisting of albumin, globulin and mixtures thereof fromblood or whey, which comprises raising the pH of the solution to atleast 8.5 by the addition of a composition consisting essentially of aphysiologically compatible basic compound at a temperature below thedenaturing temperature of the protein, decreasing the pH immediatelythereupon to the iso-electric point of the protein by the addition of acomposition consisting essentially of an acid at a temperature below thedenaturing temperature of the protein and separating the precipitatedprotein from the solution.

2. A process according to claim 1 wherein the initial protein solutioncontains a mixture of albumin and globulin 5 to 6 percent by weight.

3. A process according to claim 2 wherein the naturally occurringaqueous solution is blood.

4. A process according to claim 2 wherein the naturally occurringaqueous solution is whey.

5. A process according to claim 1, in which the precipitated albumin andglobulin is redispersed to an aqueous solution, which is used as thestarting material for repeating the process.

6. A process according to claim 1 wherein the pH is raised to 9.5-lO.5by the addition of basic compound.

7. A process according to claim 1 wherein the acid is added at 45centigrade.

8. A process according to claim 1 wherein the separation of the proteinis accomplished by passing the solution through a centrifuge.

2. A process according to claim 1 wherein the initial protein solution contains a mixture of albumin and globulin 5 to 6 percent by weight.
 3. A process according to claim 2 wherein the naturally occurring aqueous solution is blood.
 4. A process according to claim 2 wherein the naturally occurring aqueous solution is whey.
 5. A process according to claim 1, in which the precipitated albumin and globulin is redispersed to an aqueous solution, which is used as the starting material for repeating the process.
 6. A process according to claim 1 wherein the pH is raised to 9.5-10.5 by the addition of basic compound.
 7. A process according to claim 1 wherein the acid is added at 45* centigrade.
 8. A process according to claim 1 wherein the separation of the protein is accomplished by passing the solution through a centrifuge. 